Rb and N-ras Function Together to Control Differentiation in the Mouse The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Takahashi, C., R. T. Bronson, M. Socolovsky, B. Contreras, K. Y. Lee, T. Jacks, M. Noda, R. Kucherlapati, and M. E. Ewen. 2003. “Rb and N-Ras Function Together To Control Differentiation in the Mouse.” Molecular and Cellular Biology 23 (15): 5256–68. doi:10.1128/ MCB.23.15.5256-5268.2003. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41543053 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA MOLECULAR AND CELLULAR BIOLOGY, Aug. 2003, p. 5256–5268 Vol. 23, No. 15 0270-7306/03/$08.00ϩ0 DOI: 10.1128/MCB.23.15.5256–5268.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Rb and N-ras Function Together To Control Differentiation in the Mouse Chiaki Takahashi,1† Roderick T. Bronson,2,3 Merav Socolovsky,4 Bernardo Contreras,1 Kwang Youl Lee,1‡ Tyler Jacks,5 Makoto Noda,6 Raju Kucherlapati,7 and Mark E. Ewen1* Department of Medical Oncology and Medicine, Dana-Farber Cancer Institute and Harvard Medical School,1 and Rodent Histopathology Core3 and Harvard-Partners Center for Genetics and Genomics,7 Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology, Tufts University Schools of Medicine and Veterinary Medicine, 2 Boston, Massachusetts 02111 ; Whitehead Institute for Biomedical Research, Massachusetts Institute of Downloaded from Technology, Cambridge, Massachusetts 021424; Department of Biology and Howard Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 021395; and Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan6 Received 8 November 2002/Returned for modification 7 January 2003/Accepted 30 April 2003 The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote dif- ferentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control http://mcb.asm.org/ of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine em- bryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the develop- mental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb؊/؊;N-ras؊/؊ skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb؊/؊;N-ras؊/؊ muscle shows a restoration in the expression of the late muscle-specific gene ,MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb؊/؊;N-ras؊/؊ ؊/؊ ؊/؊ ؊/؊ compared to Rb myoblasts in culture. The improved differentiation of skeletal muscle in Rb ;N-ras on October 13, 2019 by guest embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable. The loss of tumor suppressor gene function is a common through its interaction with E2F, and its association with other event in the development of human cancer. The retinoblas- factors involved in altering chromatin structure (e.g., histone toma gene (Rb) has served as the paradigm for the study of this deacetylase and BRG1) leads to active repression of genes class of genes (67, 68). Mutations resulting in the inactivation involved in cell proliferation (e.g., cyclin E). The ability of pRb to of Rb are found in a large fraction of human cancers of both influence cell death also appears to involve its regulation of mesenchymal and epithelial origin (57, 62). An understanding E2F (13, 56, 63). of how the retinoblastoma protein (pRb) exerts its tumor- How pRb regulates differentiation is poorly understood. suppressive action can be gained from knowledge of the bio- Most differentiation programs involve withdrawal from the cell logical and molecular consequences of its inactivation. cycle, but the participation of pRb appears to extend beyond pRb participates in the control of cell cycle progression, an ability to merely facilitate the process by inhibiting E2F and apoptosis, and differentiation. How it exerts effects on prolif- cell cycle progression. Naturally occurring mutants of pRb can eration is well understood. This appears to be achieved by its be identified that retain tumor suppressor activity and the regulated interaction with the E2F family of transcription fac- ability to promote differentiation when assayed in vitro despite tors (14, 56, 63). E2F can bind to and promote the expression having lost the capability to bind to E2F (28, 58). Consistent of a number of genes involved in cell cycle progression (e.g., with this, pRb influences the activity of a number of transcrip- DHFR). pRb, by binding to E2F, can inhibit the transactivation tion factors known to participate in different differentiation function of E2F. In addition, pRb is targeted to promoters processes, such as MyoD, the glucocorticoid receptor, CBFA1, and C/EBP␤ (3, 4, 11, 43, 44, 59, 61). Also, cell culture studies * Corresponding author. Mailing address: Department of Medical have demonstrated a key role for pRb in myogenesis, osteo- Oncology and Medicine, Dana-Farber Cancer Institute and Harvard genic differentiation, and adipogenesis (3, 6, 11, 43, 54, 58, 61). Medical School, Boston, MA 02115. Phone: (617) 632-2206. Fax: (617) Mouse genetics have been employed to better understand 632-5417. E-mail: [email protected]. the physiological functions of pRb (35, 66). Mice heterozygous † Present addresses: Department of Molecular Oncology, Kyoto Uni- for Rb succumb to pituitary tumors (15, 17, 19), while inacti- versity Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan. ‡ Present address: Chung-buk National University, College of Med- vation of both Rb alleles results in embryos that die in mid- icine, Cheong-Ju, Korea. gestation (5, 19, 31, 71). These embryos are characterized by 5256 VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5257 defects in erythroid, neuronal, and skeletal muscle differenti- and cell death. Our analyses suggest that N-ras operates with ation, and ectopic S-phase entry and apoptosis are observed in Rb in the control of cellular differentiation. the central nervous system (CNS), peripheral nervous system (PNS), lens, and skeletal muscle (5, 19, 31, 32, 71). The con- MATERIALS AND METHODS tribution of deregulated E2F activity to these phenotypes has Mouse strains. Parental Rbϩ/Ϫ and N-rasϩ/Ϫ mice were maintained on a been assessed with compound embryos lacking Rb and E2f-1 or mixed genetic background (C57BL/6 ϫ 129/Sv and C57BL/6 ϫ 129/Ola, respec- ϩ/Ϫ ϩ/Ϫ E2f-3 (21, 64, 72). These embryos live longer than their RbϪ/Ϫ tively) and intercrossed to generate subsequent founders. Rb ;N-ras fe- males were crossed with Rbϩ/Ϫ;N-rasϩ/ϩ, Rbϩ/Ϫ;N-rasϩ/Ϫ,orRbϩ/Ϫ;N-rasϪ/Ϫ counterparts, and this has been attributed to a partial restora- males. Timed pregnancies were established by the detection of a plug, taken as tion of fetal liver erythropoiesis. Additionally, loss of either embryonic day 0.5 (E0.5). Mice and embryos were genotyped by PCR with E2f-1 or E2f-3 can suppress the deregulated proliferation and genomic DNA extracted from tails and yolk sacs, respectively, as previously apoptosis to significant but varying degrees in the CNS, PNS, described (19, 65). All animal experimentation was performed at the Dana- Farber Cancer Institute Animal Resource Facility in accordance with the guide- and lens. Importantly, the lens of Rb-deficient embryos also lines of the National Institutes of Health. shows signs of aberrant differentiation that are not rescued by Histology and immunohistochemistry. Embryos were fixed in Bouin’s solution, Downloaded from loss of E2f-1, suggesting again that Rb’s influence on cell cycle rinsed with 70% ethanol, and embedded in paraffin for sectioning. Sections (6 progression and apoptosis is genetically separable from its ␮m) were stained with hematoxylin and eosin (H&E). Alternatively, sections regulation of differentiation (36). A notable aspect of these were incubated with a monoclonal antibody (MY-32; Sigma) to myosin heavy chain (MHC) following deparaffinization and rehydration. To identify prolifer- Ϫ/Ϫ studies is the observation that the extended life span of Rb ; ating cells, bromodeoxyuridine (BrdU) was injected intraperitoneally (33 ␮g per Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ E2f-1 / and Rb / ; E2f-3 / embryos reveals additional phe- mouse) 1 h prior to sacrifice. Fixed embryos were rinsed with 70% ethanol and notypes, including developmental defects in the lung and embedded in paraffin, from which sections were cut. After rehydration, the heart, and more pronounced defects in skeletal muscle. Ab- endogenous peroxidase activity was quenched with 3% H2O2–10% methanol in phosphate-buffered saline (PBS) (pH 7.4). Sections were then treated succes- normalities in skeletal muscle differentiation are also observed sively with 0.05 mM trypsin, 2 N HCl, and PBS (pH 6.0). After blocking with 6% in Rb-deficient mice with a partial reconstitution of Rb (71) goat serum, sections were incubated with an anti-BrdU mouse monoclonal an- http://mcb.asm.org/ and in RbϪ/Ϫ; Id2Ϫ/Ϫ mice (30). Indeed, respiratory failure tibody (B44; Becton Dickinson) in the presence of 0.5% Tween 20 in PBS (pH resulting from the complete lack of muscle fibers in the dia- 7.4).